PROJECT SUMMARY/ABSTRACT (RESEARCH PROJECT II) The trophoblast lineage arises early in embryonic development through a complex interaction of transcriptional and epigenetic regulators controlling key signaling pathways. Trophoblast stem (TS) cells possess the capacity to self-renew and differentiate into specialized trophoblast cell populations required for the establishment and maintenance of pregnancy. However, the epigenetic modifications, and the hierarchy of the transcriptional regulators, which determine trophoblast lineage, maintain the trophoblast cell stem state and regulate the multilineage differentiation are poorly understood. We have identified two related proteins, special AT-rich sequence-binding protein 1 (SATB1) and SATB2, which can act as chromatin organizers and transcriptional regulators, contribute to both the maintenance and expansion of TS cells and the inhibition of trophoblast differentiation. SATB proteins are proposed to play a key role in the regulatory network controlling trophoblast development. In this research project, three specific aims are proposed: 1) to investigate the role of SATB proteins in the regulation of trophoblast lineage determination; 2) to establish the position of SATB proteins in the hierarchy of regulators controlling the TS cell stem state; 3) to evaluate the in vivo role of SATB proteins in trophoblast development. The proposed study will utilize in vitro rodent and human stem cell models and mouse mutagenesis to explore the role of SATB proteins in regulating the trophoblast development. Genome wide analysis of transcriptional and epigenetic modifications will be coupled to transcriptome analysis to identify upstream regulators of SATB expression and downstream targets of SATB action. This research investigation will reveal novel properties of trophoblast stem cell regulation and expand our understanding of the disorders associated with early pregnancy loss.